Project Summary The ability to preserve the female gamete (oocyte) is critical for women wanting to retain fertility before cancer therapy, treat infertility or delay reproduction. However, oocyte freezing is complex because of the cell?s large size, significant water content and fragile cellular skeleton. Storing this cell and other biomaterials at ultra-low temperature also is costly, which complicates the USA-wide need for more biorepositories to address human health issues. Our research is advancing a transformative way of preserving the maternal genome by compacting, drying, storing at supra-zero temperatures and reanimating the oocyte?s nucleus (germinal vesicle, GV) rather than the entire cell. We study the domestic cat, a model that shares many physiological mechanisms and genomic similarities with the human. In the recent years, we have demonstrated that: 1) good quality oocytes can be reconstructed with competent GVs from early stage follicles that are delivered into high quality, recipient cytoplasts; 2) fundamental epigenetic marks and key proteins of the GV are controlled and sustained within the nucleus, thereby confirming the value of preserving isolated nuclei; and 3) desiccated cat GVs retain viability for weeks, with the remaining challenge being to maintain a low and stable moisture content to prevent degradations during storage. Findings to date have guided us to three priorities: 1) desiccation of the GV to an intermediate moisture level combined with biostabilizing agents; 2) a precisely formulated rehydration protocol; and 3) production of embryos and viable offspring with unaltered epigenetic patterns. The overall hypotheses are: 1) the maternal genome and associated nuclear factors are securely preserved at room temperature by desiccating the GV at a higher (more accommodating) moisture content when combined with biostabilizing agents; 2) key GV components are fully recovered by an appropriate rehydration process after storage; and 3) these enhancements promote GV survival and reanimation in a recipient cytoplast as well as the ability of the reconstructed oocyte to mature, be fertilized and successfully develop into a normal embryo in vitro/in vivo. Likelihood of achieving the project?s priorities is high given our recent results and our access to novel, advanced tools, including new controlled environmental chambers for the desiccation and Tubular Perfusion Systems for rehydration as well as computational transcriptomics/epigenomics to validate the viability of resulting embryos. Besides providing an improved understanding of the resilience of the oocyte?s nucleus, end products will inform on the practicality and safety of GV storage in reproductive health care, management of biomedical models and alternative options in building lower- cost repositories.